Further insights on the secondary metabolites of a soft coral-derived fungus Aspergillus versicolorunder the guidance of MS/MS-based molecular networking led to the isolation of seven known cycloheptapeptides, namely, asperversiamides A–C (1–3) and asperheptatides A–D (4–7) and an unusual pyrroloindoline-containing new cycloheptapeptide, asperpyrroindotide A (8). The structure of 8was elucidated by comprehensive spectroscopic data analysis, and its absolute configuration was determined by advanced Marfey’s method. The semisynthetic transformation of 1into 8was successfully achieved and the reaction conditions were optimized. Additionally, a series of new derivatives (10−19) of asperversiamide A (1) was semi-synthesized and their anti-tubercular activities were evaluated against Mycobacterium tuberculosisH37Ra. The preliminary structure−activity relationships revealed that the serine hydroxy groups and the tryptophan residue are important to the activity.