<p>Antibiotics (ATBs) remain a cornerstone in the treatment of bacterial infections, yet clinical practice increasingly incorporates photonic therapies such as photobiomodulation (PBM) and photodynamic therapy (PDT). These modalities, widely used in infectious and inflammatory conditions, are frequently administered concomitantly with ATBs. However, the potential photochemical interactions between β-lactam ATBs and light-based therapies remain poorly understood, raising questions about efficacy and safety in combined treatment settings. In this study, we characterized the optical and photochemical properties of representative β-lactam ATBs, including penicillins (amoxicillin, oxacillin), cephalosporins (cephalothin, cefazolin, ceftazidime, ceftriaxone, cefuroxime), and a carbapenem (meropenem). Using diffuse transmittance and reflectance spectroscopy, we calculated absorption and scattering coefficients via the Kubelka–Munk function. Reactive oxygen species (ROS) generation was assessed under direct blue light (460&#xa0;nm) illumination and red light exposure in the presence of methylene blue (MB), a clinically employed photosensitizer. Cephalosporins demonstrated significant ROS generation under blue light, whereas penicillins and meropenem did not. In PDT-like conditions, meropenem enhanced MB-mediated ROS production and promoted MB photobleaching, suggesting potentiation of PDT effects. In contrast, penicillins suppressed MB-driven ROS generation, potentially limiting PDT efficacy. Cephalosporins showed heterogeneous effects, either enhancing, reducing, or leaving MB-induced ROS unchanged. These findings indicate that β-lactam ATBs exert distinct photobiological behaviors with potential clinical consequences. Cephalosporins may act as intrinsic photosensitizers, meropenem may potentiate PDT, and penicillins may attenuate phototherapy efficacy. Recognizing such interactions is critical to optimizing combined ATB–phototherapy regimens and avoiding unintended antagonism or toxicity in clinical practice.</p>