Background: Periprosthetic joint infections (PJI) are among the most morbid complications in total hip arthroplasty (THA). The ideal incubation time, however, for intraoperative cultures for PJI diagnosis remains unclear. As such, the aim of this study was to determine if any differences existed in culture-positive rates and organism detection between five-day and fourteen-day cultures. Methods: This retrospective cohort study consisted of THA cases diagnosed with PJI performed between May 2014 and May 2020 at a single tertiary-care institution. Analyses compared five-day and fourteen-day cultures and carried out a pre-specified subgroup analysis by organism and PJI type. Results: A total of 147 surgeries were performed in 101 patients (57.1% females), of which 65% (n = 98) obtained five-day cultures and 34% (n = 49) obtained fourteen-day cultures. The positive culture rate was 67.3% (n = 99) with Staphylococcus aureus being the most common pathogen identified (n = 41 specimens, 41.4%). The positive culture rate was not significantly different between groups (66.3% five-day, 69.4% fourteen-day, p = 0.852). Fourteen-day cultures had a significantly longer time-to-positive culture (5.0 days) than five-day cultures (3.0 days, p < 0.001), a higher rate of fungi (5.6% vs. 0%), and a lower rate of Gram-negatives (4.5% vs. 18.7%, p = 0.016). Conclusions: Fourteen-day cultures did not increase the positivity rate, had higher rates of slow-growth pathogens, and had a longer time-to-positivization than five-day cultures. Prolonged culture holds may provide more thorough organism detection for PJI without increasing the diagnostic culture yield.