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The increasing incidence of periprosthetic joint infections (PJIs) has led to a growing interest in developing strategies to prevent and treat this severe complication. The surgical site’s application of antiseptic solutions to eliminate contaminating bacteria and eradicate the bacterial biofilm has been increasing over time. Even though it has been proven that combining antimicrobials could enhance their activities and help overcome acquired microbial resistance related to the topical use of antibiotics, the toxicity of integrated solutions is not well described. This study aimed to evaluate the cytotoxicity of solutions containing povidone–iodine (PI) and hydrogen peroxide (H2O2), alone or in combination, after 1.3 and 5 min of exposure. Chondrocytes, tenocytes, and fibroblast-like synoviocytes were used for cytotoxicity analysis. Trypan blue stain (0.4% in PBS) was applied to evaluate the dead cells. All solutions tested showed a progressive increase in toxicity as exposure time increased except for PI at 0.3%, which exhibited the lowest toxicity. The combined solutions reported a reduced cellular killing at 3 and 5 min than H2O2 at equal concentrations, similar results to PI solutions.