Two new steroid derivatives [Withaperuvin O: (20S, 22R)−5α, 6β, 14α, 17β, 20α-pentahydroxy-1-oxowithan-24-en-22,26-olide (1) and Withaperuvinoside: (24E, 22R)−1α,3β-dihydroxyergosta-5,24-dienolide-26-O-β-_D-glucopyranosyl (1→2)-β-_D-glucopyrano-side] (2) were isolated from the methanolic extract of the whole plant of Physalis peruviana together with six known compounds: saringosterol (3), physalindicanol B (4), physalindicanol A (5), perulactone (6), β-sitosterol (7), and β-sitosterol-3-O-β-_D-glucopyranoside (8). Their structures were established on the basis of spectroscopic (1D and 2D NMR) and spectrometric evidence and by comparison with data described in the literature. Compounds 1 and 2 are closely related to compounds previously isolated from the same plant. The antibacterial activity of the crude extract, EtOAc and n-BuOH fractions as well as compounds 1-6 was assessed by determining the minimum inhibitory concentration (MIC) using the broth microdilution method against five pathogenic bacterial strains. The methanolic extract and the EtOAc fraction showed a weak activity against all the tested bacterial strains with MICs values between 128 and 512 µg/mL, except for Staphylococcus aureus. Compounds 1 and 2 were inactive against the tested microorganisms.