The study of the acetone and methanol extracts of the stems, leaves, and flowers of Physalis volubilis led to obtain four withanolide glycosides (4, 6, 10, and 14). Compounds 1-3, 13, 15, and a sterol glycoside were also isolated. The structures were established by analysis of spectroscopic and spectrometric data, and by chemical transformations. Nine of the obtained compounds (2, 3, 6, 7, 10, 11, and 13-15) were evaluated for their qualitative antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. Compound 2 showed the highest inhibition of bacterial growth. Its minimum inhibitory concentrations against S. aureus ATCC® 25923™ and a clinical isolate of Methicillin-resistant S. aureus (MRSA) were 62.5 and 125 μg/mL, respectively.