Background: Nontuberculous mycobacterial (NTM) lung infections are a major public health concern. Diagnosis of NTM-pulmonary disease (NTM-PD) is difficult because its clinical, microbiological, and radiological features resemble to those of pulmonary tuberculosis (TB), leading to misdiagnosis. Identification at the species level is essential for diagnosis and determination of therapy, which is currently not performed routinely in Indonesian laboratories. Methodology and principal findings: From January 2020 to May 2021, 94 NTM isolates were collected from three TB referral centers in Java Province. Species were identified using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tests were performed to determine antibiotic susceptibility, biofilm formation ability, sliding motility characteristics, and the ability to adhere to and invade pneumocytes. After identifying the species of all the isolates, we found nine groups of NTMs: M. fortuitum group 51% (48/94), M. abscessus 38.3% (36/94), M. intracellulare 3.1% (3/94), M. neoaurum 2.1% (2/94), M. chelonae 1.1% (1/94), M. gordonae 1.1% (1/94), M. szulgai 1.1% (1/94), M. mucogenicum 1.1% (1/94), and M. arupense 1.1% (1/94). Amikacin was the most effective antibiotic against M. fortuitum group and M. abscessus. The M. fortuitum group was significantly better at forming biofilms than M. abscessus, but both had the same sliding motility capability. The ability of the M. fortuitum group to adhere to and invade pneumocytes was better than that of M. abscessus, with the number isolates of the M. fortuitum group capable of superior adhesion and invasion to that of M. abscessus. Conclusions/Significance: This study shows that M. fortuitum group and M. abscessus were the most common NTM found in Java, Indonesia. The M. fortuitum group and M. abscessus were the most susceptible to amikacin; therefore, this was the empirical treatment of choice. The ability to form biofilms is directly proportional to the ability to adhere to and invade pneumocytes but not to the susceptibility profile or sliding motility characteristics. Author summary: Nontuberculous mycobacterial (NTM) infection is a serious public health problem that can cause various clinical manifestations of infection, including NTM-pulmonary lung disease (NTM-PD). NTM-PD infections are often misdiagnosed as pulmonary tuberculosis, leading to underestimation of the incidence of NTM infections, particularly in countries where tuberculosis is endemic. The lack of routine laboratory services in Indonesia in terms of species identification and susceptibility to NTM is a factor complicating diagnosis and patient management. These factors have led to a lack of adequate information on NTM infections in Indonesia. Research on species identification and susceptibility testing is important to help clinicians better manage NTM infections. This study was conducted on 94 clinical isolates of NTM from Java Province, and it is the first study in Indonesia to identify NTM to the species level using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The most common species found were M. fortuitum group and M. abscessus. M. abscessus was more resistant to ciprofloxacin, moxifloxacin, amikacin, trimethoprim/sulfamethoxazole, and doxycycline than M. fortuitum group. Amikacin is the most effective antibiotic and can be used as the empirical antibiotic of choice. [ABSTRACT FROM AUTHOR]