Staphylococcus aureus is a widespread pathogen in nature, with staphylococcal enterotoxins being a major cause of foodborne illness. The extensive use of antibiotics on farms has contributed to the spread of antibiotic-resistant S. aureus. Understanding the fitness cost and compensatory evolution of antibiotic-resistant isolates is crucial for assessing the consequences of resistance acquisition and predicting the potential spread of resistant mutants in various environments. In this study, penicillin (PEN) was used to induce resistance in antibiotic-sensitive S. aureus, resulting in PEN-resistant mutants. We evaluated and compared the growth and thermal inactivation characteristics at different temperatures, virulence potential, and relative fitness of S. aureus isolates before and after PEN exposure under various stress conditions. The results revealed that PEN induction led to the acquisition of multidrug resistance and cross-resistance in S. aureus. Compared to the parent sensitive isolates, PEN-resistant S. aureus exhibited altered biological characteristics, including reduced phenotypes related to invasion (hemolysis activity, serum resistance) and toxin production (staphyloxanthin formation), but increased characteristics linked to colonization (biofilm formation) and gene transfer (autolytic activity). Fitness advantages were either maintained or enhanced in resistant isolates, with PEN serial passaging showing a more pronounced effect in improving fitness and driving compensatory evolution. These findings underscore the importance of investigating fitness costs and compensatory evolution following resistance acquisition to better understand the risks posed by resistant S. aureus to the food chain and human health.