Cefazolin is an essential antibiotic used for the treatment of bacteremia; in particular, it is recommended as a first-line agent for infections caused by methicillin-susceptible Staphylococcus aureus (MSSA). In March 2019, problems with a major antibiotic supplier caused a critical shortage of cefazolin in Japan. In fact, in our hospital, the prescription of cefazolin was restricted (unless permitted by an infectious disease physician) between March 2019 and January 2020. The aim of this study was to evaluate the clinical outcomes of patients with MSSA bacteremia at a university hospital in Japan during cefazolin shortage. Details of antimicrobial use and patient data were extracted from the medical records and classified as pre-shortage (March 2018–January 2019) and post-shortage (March 2019–January 2020). Seventy-five patients were included in the study (pre-shortage group, n = 39; post-shortage group, n = 36); there were no significant differences between the demographic characteristics of the two groups. The percentage of patients that received cefazolin as definitive therapy, was significantly lower in the post-shortage group than that in the pre-shortage group (82% vs. 53%, p = 0.014). Of note, penicillins (including benzylpenicillin, ampicillin, ampicillin/sulbactam, and piperacillin/tazobactam) were more frequently administered as alternative therapy to the post-shortage group (10% vs. 53%, p < 0.001). However, no significant differences were observed between the following clinical outcomes of the two groups: total duration of antibiotic therapy, time to fever resolution and white blood cell normalization, time to negative blood cultures, length of hospitalization after the onset of bacteremia, treatment failure, 30-day mortality, and adverse drug reactions (diarrhea and skin rash). Therefore, in the treatment of MSSA-induced bacteremia, cefazolin shortage was associated with an increase in the use of penicillins as alternative agents, with no major changes in the clinical outcomes.