Introduction Surgical site infections (SSIs) with methicillin-resistant Staphylococcus aureus are serious complications of spinal instrumentation surgery. Many spine surgeons are concerned that using prophylactic vancomycin powder will lead to certain risks: the development of multidrug-resistant pathogens, anaphylactic reactions, and organ toxicity. Minimally invasive spine stabilization (MISt) is associated with shorter operation times and less blood loss and may therefore require the use of less vancomycin powder, which may reduce these risks. This retrospective comparative study of patients who underwent MISt at a single institution aimed to evaluate the complications (such as allergy, SSIs, and organ toxicity) and the local and serum levels associated with using prophylactic intrawound vancomycin powder compared with IV cefazolin alone. Methods Thirty-four patients received intrawound vancomycin powder (1 g) applied during wound closure in minimally invasive posterior lumbar interbody fusion (MIS-PLIF). This group was compared with 133 control patients who did not receive vancomycin. White blood cell counts and C-reactive protein (CRP) levels were measured for both groups on postoperative days (PODs) 1, 3, and 7 and were statistically analyzed. In the vancomycin group, serum vancomycin levels were measured on PODs 1, 3, 7, and 14; drain vancomycin levels and postoperative blood loss were determined on PODs 1 and 2. Results The CRP levels on PODs 1 and 3 were significantly higher in the vancomycin group than in the control group (P<0.001, P=0.024). In the vancomycin group, mean drain levels trended downward from 313 μg/mL (POD 1) to 155 μg/mL (POD 2). These levels correlated negatively with drain drainage volume on both days (POD 1: r=-0.48, P=0.015; POD 2: r=-0.47, P=0.019). Mean serum vancomycin levels also trended downward from 2.3 μg/mL (POD 1) to 1.7 μg/mL (POD 14). Conclusions Our results unexpectedly demonstrated that the local application of vancomycin powder causes an acute inflammatory response and the long-term detection of low serum vancomycin levels. Less than 1 g of intrawound vancomycin powder may be useful only at high risk of SSI.