Interest in medicinal and aromatic plants as a source of antimicrobial drugs has emerged, mainly due to increased resistance of infectious agents. In this study, the antifungal and cytotoxic activity of thirteen essential oils and nine extracts of plants of the genus Piper, from Colombia, were evaluated. The Piper hispidum Sw oil showed activity against the multi-resistant fungus Fusarium oxysporum. Six samples showed strong activity against dermatophytes, being Piper bogotense the most active, followed by P. hispidum. The oils from Piper hispidum, Piper coruscans, Piper marginatum and Piper cf. divaricatum, and the extract from Piper sanctifelicis, were not cytotoxic, unlike Piper bredemeyeri, Piper eriopodon and Piper bogotense oils, which exhibited significant cytotoxicity. The main components of the most active samples showed the presence of trans-β-caryophyllene. In addition, caryophyllene oxide, β-pinene and α-pinene were present in most antifungal oils at different concentrations. These results confirm the anti-mycotic potential of essential oils of the Piper genus against the most pathogenic fungi, as well as against important dermatological fungi.