The indiscriminate use of antibiotics has led to the increase in antibiotic-resistant microorganisms, particularly the agents of high-morbidity diseases. Even though some studies report the use of bryophytes as sources of antimicrobial compounds, further research is necessary to elucidate the existence and potentialities of such bioactive compounds, especially in Brazil. This study investigated the antimicrobial properties of the ethanolic extract of Orthostichella rigida species of music, collected at different times of the year against bacteria and fungi that may be human pathogenic, the first of food origin and the second as opportunistic. Antimicrobial activity was verified using the broth microdilution method (MIC-MBC/MFC), assayed in triplicate. Seasonal influence was determined from the absolute values of MIC and MBC/MFC together with the triplicate average of these same methods. O. rigida ethanolic extract was effective in antimicrobial analysis for all microorganisms tested, being that the species less sensitive to O. rigida extracts was the fungus Candida albicans when compared to the other isolates tested. The highest sensitivity to O. rigida extracts were for Listeria monocytogenes (19.53 μg/mL), Cryptococcus neoformans (39.06 μg/mL), Staphylococcus aureus and Salmonella enteritidis (78.13 μg/mL). The extracts from the summer, autumn and winter seasons were more efficient to reach MIC, CBM and CFM, with values below 100 μg/mL for both, which is considered potent. Seasonal influence is statistically evident in spring, which has demonstrated minor antimicrobial activity when compared with the other seasons. Mosses are understudied regarding their compounds and O. rigida has demonstrated a rising potential for future research and possible use as a natural antimicrobial.