Due to the biological properties associated to the Baccharis genus and the limited information on its antimicrobial properties, the present work investigates the chemical composition and antibacterial, antifungal and antiprotozoal activities of the essential oils of two species, B. uncinella from Campo Alegre and B. semiserrata from Atalanta, both places located in Santa Catarina State, Southern Brazil. Oils were obtained by hydrodistillation and were analyzed by gas chromatography–flame ionization detector (GC–FID) and GC–mass spectrometry (GC–MS). The dry leaf essential oil yield from B. uncinella was 0.65% (m/m), and 0.50% and 0.22% (m/m) for dry leaf and twig from B. semiserrata, respectively. The main components in the oil from B. uncinella were α-pinene (9.0%), β-pinene (9.2%), limonene (11.5%), β-caryophyllene (13.2%) and spathulenol (9.3%). The following major compounds were identified in the leaf essential oil from B. semiserrata: α-pinene (4.3%), β-pinene (11.4%), limonene (6.7%), β-caryophyllene (8.2%), γ-muurolene (7.3%), bicyclogermacrene (8.0%), E-nerolidol (9.6%) and spathulenol (9.8%). Similarly, in the essential oil from the twigs were α-pinene (3.3%), β-pinene (7.9%), limonene (9.1%), β-caryophyllene (3.9%), bicyclogermacrene (6.0%), spathulenol (25.1%), caryophyllene oxide (8.0%) and globulol (5.6%). The antibacterial activity was determined by microdilution method. Baccharis uncinella oil was inactive for all bacteria tested and B. semiserrata twigs oil presented moderate activity against Staphylococcus aureus and the leaves weak activity against S. aureus and Bacillus cereus. The antifungal activity was determinate by the agar dilution method. Baccharis uncinella oil was active against M. gypseum, T. mentagrophytes, C. neoformans and M. canis. Both, leaf and twig essential oils from B. semiserrata were active against Microsporum gypseum, Candida albicans, Epidermophyton flocosum, Trichophyton mentagrophytes and Cryptococcus neoformans. Baccharis semiserrata leaf essential oil was also active against Trichophyton rubrum. In the antiprotozoal assays, leaf essential oil from B. uncinella gave an IC50 of 223 μg/mL against Tripanossoma cruzi. Both leaf essential oils were active only at 500 μg/mL against Leishmania braziliensis.