Drug-resistant organisms (DROs) necessitate the development of new therapies. Antimicrobial blue light (ABL) is a promising option, utilizing photoexcitation of endogenous bacterial components to generate reactive oxygen species, leading to bacterial death. The aim of this study is to investigate the effects of a novel isotropic optical fiber under in-vitro conditions on multidrug-resistant gram-negative Pseudomonas aeruginosa (MDR-Pa) and methicillin-resistant Staphylococcus aureus (MRSA). Time-to-kill assays were conducted in tubes containing 10 mL of 0.9% NaCl solution with an inoculum of 1 × 10⁵ CFU/mL for MDR-Pa or MRSA. The experiments were repeated at least three times per strain. Experimental tubes had either one (low power, LP) or two (high power, HP) optical fibers delivering five ABL wavelengths (405, 415, 435, 450, and 475 nm) over 60 min. Control tubes lacked optical fibers. Samples were taken at 0, 10, 20, 30, and 60 min, streaked on agar, and incubated to determine CFU/mL. Bactericidal reduction was defined as a ≥ 99.9% (≥ 3 log₁₀) reduction in CFU/mL. One-way ANOVA were conducted. The novel isotropic optical fiber was able to exhibit bactericidal effects for MDR-Pa only under HP-ABL with a log₁₀CFU/mL ± SD difference of −3.71 ± 0.01 at 60 min (p = 0.03). Conversely, the optical fiber exhibited bactericidal effects on MRSA under both LP-ABL and HP-ABL with a log₁₀CFU/mL±SD difference of −3.73 ± 0.08 at 60 min (p = 0.03) and −3.07 ± 0.28 at 20 min (p = 0.02), respectively. The isotropic optical fiber demonstrated bactericidal effects on MRSA and MDR-Pa in in-vitro studies and shows potential as a therapeutic option for DROs.