Medicinal herbs play a major role in therapeutics due to phytochemicals and can be alternatives to chemical drugs. Physalis minima fruit exhibits multiple biological activities owing to vitamins, minerals, and antioxidants. This work investigated phytochemical variations of genetic variants from four districts in India using PCR and RAPD methods. Phytochemicals were analyzed via thin layer chromatography with chloroform, diethyl ether, ethanol, ethyl acetate, and methanol extracts. Ethanol crude extract was characterized by FT-IR and GC-MS. Antimicrobial activity of ethanolic extracts was evaluated against Streptococci acidominimus, Pseudomonas aeruginosa, Penicillium expansum, and Aspergillus fumigatus via cup diffusion method. Inhibition zones ranged 1.5–5 mm for bacteria and 0.5–2.18 mm for fungi. RAPD analysis confirmed phenotypic and genotypic characteristics. © 2022 Elsevier B.V., All rights reserved.