Background: Bacterial and fungal infections are an emerging public health concern, especially in the context of nosocomial infections. This study discusses the use of peppermint (Mentha piperita L.) methanolic extract against bacterial strains such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus and fungal species such as Trichophyton mentagrophytes, Trichophyton verrucosum, and Candida albicans. Objectives: This research aimed to investigate the inhibitory potential of M. piperita methanolic extract against some bacteria and fungi isolated from humans. Materials and Methods: The methanolic extract was prepared by standard procedure, and total phenol, total flavonoid, and total anthocyanin content were determined using a spectrophotometer. Antimicrobial activity against the aforementioned bacteria and fungi strains was evaluated using the disc diffusion method. Results: The contents of total phenol, flavonoid, and anthocyanin as well as antimicrobial activities and antifungal properties were enhanced by the increase of extract concentration. The results showed that the antimicrobial activity of leaf extract was more powerful against Bacillus subtilis (1.39 mm) than Staphylococcus aureus (1.29 mm), Escherichia coli (1.20 mm), and Pseudomonas aeruginosa (1.10 mm) based on the growth inhabitation zone. The most powerful antifungal activity of peppermint extract was observed against Trichophyton verrucosum and Trichophyton mentagrophytes (1.49 and 1.48 mm, respectively). Conclusions: Due to phenolic content, methanolic extract of M. piperita has moderate antibacterial and antifungal properties against the aforementioned gram positive and gram negative bacteria and fungi strains.