Bovine milk is a nutritionally rich fluid containing bioactive proteins that support immune function and growth. Traditional thermal pasteurization (72°C for >15 s) ensures microbial safety but degrades heat-sensitive proteins. High-pressure processing (HPP) offers a nonthermal alternative for microbial reduction, yet its effect on protein structure, particularly under applications of multiple pressure cycles, remains underexplored. This study aimed to evaluate the effectiveness of single- and dual-cycle HPP treatments for bacterial inactivation and protein preservation in whole bovine milk and to compare these results with the industry standard—high-temperature, short-time (HTST) processing. Raw bovine milk samples were inoculated with vegetative pathogens (Listeria monocytogenes, Staphylococcus aureus) or spores (Bacillus cereus, Bacillus subtilis) and treated with varying HPP conditions (350–600 MPa; 4–12 min, at 30°C, for single or dual cycles). Microbial reduction was assessed by standard plate count. Whey protein retention (lactoferrin [LF], IgA, IgG, IgM) was quantified using ELISA and compared with HTST and raw milk controls. Dual-cycle HPP treatments significantly enhanced bacterial reduction compared with single-cycle time equivalents for S. aureus and B. subtilis, but not for L. monocytogenes or B. cereus. Treatments for S. aureus demonstrated 0.6 to 2.5 log reduction increases from single to dual cycles at pressures of 350 to 600 MPa. Although no tested treatments achieved >5-log reductions in sporulated B. subtilis, dual-cycles increased reductions by 1.2 log compared with single-cycle time equivalents. Several conditions achieved >5-log reductions for vegetative pathogens, including 600 MPa, 12 min, single cycle; 550 and 600 MPa, 4 min dual cycle, and 550 and 600 MPa, 6 min, dual-cycle. However, all HPP treatments led to substantial degradation of immunological proteins, particularly LF (53%–84% reduction), IgA (86%–95% reduction), and IgM (81%–98% reduction), with protein retention decreasing as pressure and cycle time increased. High-temperature, short-time processing preserved higher levels of native protein structure across all treatments.