Abstract Antimicrobial, antimycobacterial and antiviral activities of crude extracts, fractions and subfractions of Tabernaemontana catharinensis were evaluated. Best antimicrobial results occurred with the dichloromethane (DCM) and butanolic (NB) fractions against Staphylococcus aureus, Micrococcus sp., Enterococcus faecalis and Bacillus subtilis (minimal inhibitory concentration, MIC=31.25–1000μg/mL). Considering the Gram-negative bacteria, only NB fraction was effective against Proteus mirabilis and Aeromonas sp. (MIC=62.5μg/mL and 250μg/mL, respectively). In addition, the fungi Candida albicans, Candida glabrata, Cryptococcus neoformans, Saccharomyces cerevisiae, Aspergillus flavus and Aspergillus fumigatus were particularly vulnerable for DCM fraction (MIC=31.25–1000μg/mL). The fractions and subfractions were effective against Mycobacterium smegmatis (MIC=19.53–156.25μg/mL). DCM (selectivity index – SI=77.92), ethyl acetate (EA) (SI=40.27) and NB (SI=28.97) fractions from the leaves exhibited a potential antiviral activity toward Herpes Simplex Virus type 1 whereas DCM2 subfraction from leaves (SI=12.28) and alkaloidal fraction (AF) (10.71) maintained this good activity. Steroids, terpenoids and phenolics compounds were identified by high-performance liquid chromatography with diode array detection (HPLC/DAD) and may be partially responsible for the antimicrobial and antiherpes activities observed. The results obtained in this study showed that T. catharinensis has antimicrobial and anti-herpetic activities and that these properties are reported for the first time for this species.