Bacterial spores cause major quality and shelf life issues in fluid milk and other dairy products. Spores can only be destroyed at sterilization temperatures, which have negative effects on product quality and taste. Alternatively, bactofugation can be used as a nonthermal method to physically remove spores from milk. The objective of this work was to evaluate the impact of centrifugal force and temperature on spore removal from milk, using Bacillus licheniformis and Geobacillus spp. as challenge microorganisms. Whole raw milk was inoculated with 10⁶ to 10⁷ cfu/mL spores and centrifuged for 15 min in a lab centrifuge at g-forces representative of cream separation (2,000 × g) and bactofugation (10,000 × g). Experiments were conducted at 4°C and 50°C. Uninoculated whole milk was used as a control. Cream, skim, and pellet were collected aseptically. All fractions and the whole milk were analyzed for total aerobic bacteria (TPC), mesophilic spores (MSC), thermophilic spores (TSC) and somatic cells (SCC). A plant study was completed at a dairy plant in NY State, which has a one-phase bactofuge installed before warm (60–65°C) separation. Samples of whole raw milk, bactofuged raw milk, skim, cream, and pasteurized milk were analyzed for TPC, MSC, SCC, and psychrotrophic spores (PSC). Experiments were completed at least in triplicate, and statistical differences among means were evaluated using ANOVA at 0.05 significance level. TPC, MSC, TSC, and SCC were significantly reduced in skim by centrifugation at 4°C, regardless of centrifugal force, while bacteria and occasionally SCC significantly increased in cream. At 10,000 × g and 50°C, only TPC and TSC were significantly reduced in skim. No differences between whole, skim, or cream were observed at 2,000 × g and 50°C. In the plant trials, bactofuged whole, skim, and pasteurized milk had significantly lower TPC and SCC. MSC and PSC were reduced below detection level by centrifugation. Overall, the effectiveness of bacterial removal was primarily affected by centrifugation temperature, rather than centrifugal force. This data provides useful information about the effectiveness of centrifugation for spore removal from milk, which can positively impact dairy product quality and shelf life.