Microorganisms are ubiquitous in natural environments and in water supply infrastructure including groundwater wells. Sessile-state microorganisms may build up on well surfaces as biofilms and, if excessive, cause biofouling that reduces well productivity and water quality. Conditions can be improved using biocides and other traditional well rehabilitation measures; however, biofilm regrowth is inevitable given the continuous introduction of microorganisms from the surrounding environment. Alternative and less invasive well maintenance approaches are desirable for reducing biofilm densities while also minimizing harmful disinfection-by-products. The primary objective of this research was to evaluate effectiveness of alternative treatments for inactivating microorganisms comprising biofilms. A novel approach was designed for in situ growth of biofilms on steel coupons suspended from ‘chandeliers’. After more than 100 days of in situ growth, biofilms were harvested, sampled, and baseline biofilm densities quantified through cultivation. Ultraviolet-C (UV-C) and oxidative treatments including hydrogen peroxide (H O ), ozone (O ) and mixed oxidants were then applied to the biofilms in laboratory-scale treatments. Microbial inactivation was assessed by comparing treated versus baseline biofilm densities. H O was the most effective treatment, and decreased density below baseline by as much as 3.1 orders of magnitude. Mixed oxidants were effective for the well having a lower density biofilm, decreasing density below baseline by as much as 1.4 orders of magnitude. Disparity in the response to treatment was apparent in the wells despite their spatial proximity and common aquifer source, which suggests that microbiological communities are more heterogeneous than the natural media from which they originate.