Purpose Time-dependent surgical instrument contamination and the effect of covering during arthroplasty have not been investigated. This study aimed to evaluate time-dependent contamination of surgical instruments and the effect of covering on contamination as well as to perform bacterial typing of contaminated samples. The hypothesis was that covering the surgical instruments would decrease contamination rates. Methods Sixty patients who underwent total knee arthroplasty were randomized and divided into two groups: surgical instruments covered with a sterile towel or surgical instruments left uncovered. K-wires were used to extract microbiological samples. The K-wires were placed in a liquid culture medium at 0, 15, 30, 60, 90, and 120 min. After 24-h incubation period, samples from liquid cultures were cultured on blood agar using swabs. Samples with growth after 48 h were considered contaminated. Microscopic, staining, and biochemical properties were used for bacterial typing. Results Bacterial growth started after 30 and 60 min in the uncovered and covered groups, respectively. An increase in the number of K-wires contaminated with time was detected. At least 10,000 CFU/mL bacterial load was observed in the culture samples. Contamination was more significant in the uncovered group. A statistically significant difference in contamination was found between the uncovered and covered groups at 30-, 60-, 90-, and 120 min (p = 0.035, p = 0.012, p = 0.024, and p = 0.037, respectively). The most common bacteria on the contaminated instruments were coagulase-negative Staphylococci (60.4%), Staphylococcus aureus (22.9%), and Streptococcus agalactia (16.7%), respectively. Conclusion The risk of contamination increases with time. However, it may decrease if surgical instruments are covered. In the clinical practice, empiric antibiotic regimens based on the type of identified microorganisms in this study may be developed for postoperative periprosthetic joint infection prophylaxis.