In autumn 2016, symptoms of cobweb disease were observed on cultivated Pleurotus ostreatuscrops in Spain. Based on morphological and genetic analyses, the causal agent of cobweb was identified as Cladobotryum mycophilum. Two cropping trials, inoculated with C. mycophilum, were set up to evaluate the pathogenicity of this causal agent of cobweb. Two different inoculation methods were used: (i) an agar plug was taken from the growing edge of a C. mycophilumisolate and placed in the centre of each hole in the block of P. ostreatussubstrate (IP), and (ii) spraying each hole with a conidial suspension (ISC). In both trials, there were significant differences in disease incidence between the controls and the inoculated samples, but there were no significant differences between the two inoculation treatments. Between 75 and 87.5% of the blocks of the IP treatments and 100% of the blocks of the ISC treatments showed cobweb symptoms. Cladobotryum mycophilumwas consistently re-isolated from the inoculated blocks (100%). These findings suggest that C. mycophilumcan equally cause cobweb disease in A. bisporus, P. eryngii, and P. ostreatusmushroom crops.