Pasteurized sports drinks and other fruit-based beverages are susceptible to deterioration due to thermal processing ineffectiveness to inactivate certain spoilage microorganisms, like Alicyclobacillus acidoterrestris. This represents a major challenge for the beverage industry. The goals of this study were to: i) investigate the UV-C inactivation (annular thin film unit, actinometrical delivered fluence: 795–1270 mJ/cm2, 10–15 min, 20 °C, 1.8 L/h, Reh = 391–1067, recirculation mode operation) and the evolution during refrigerated storage of A. acidoterrestris ATCC 49025 spores and single or composite Escherichia coli ATCC 25922 in isotonic sports drinks (ISDs) made from orange (orange-ISD, UVT% = 81) or orange-banana-mango-kiwi-strawberry-lemon juices (multi-fruit-ISD, UVT% = 91), compared to a turbid orange-tangerine juice (OT juice, UVT% = 40); ii) assess the effect of pH, °Brix, A254nm, turbidity, colour and particle size of the ISDs and juice on microbial inactivation, iii) evaluate the evolution of native microbiota during cold storage, iv) investigate the Coroller, biphasic, Weibull, and Weibull-plus-tail models' ability to describe microbial inactivation and v) measure 5-hydroxymethylfurfural (HMF) formation. The modified biodosimetry method was used to calculate the germicidal UV-C fluences. Heat pasteurization (T-coil, 80 °C/6 min) was evaluated as the control treatment. UV-C was highly effective at inactivating E. coli as 4.1–5.1 and 4.5–5.6 log reductions were determined in the multi-fruit-ISD and orange-ISD, respectively, barely impacted by the background microbiota. No significant differences were recorded for the inactivation of E. coli in the UV-C and T-coil systems. Whereas, a significantly higher inactivation of A. acidoterrestris spores was achieved by UV-C (3.7–4.0 log reductions), compared to the negligible one achieved by the thermal treatment. Even though E. coli inactivation curves were similar in shape, UV-C was less effective when a cocktail of other E. coli strains was present. In comparison to the OT juice, the ISDs' inactivation kinetics were markedly different in shape, with a rapid decrease in population during the first minutes of treatment. The germicidal fluence (Hd biod) corresponding to A. acidoterrestris (19.1 mJ/cm2) was selected as it was higher than the one obtained for E. coli (11.0 mJ/cm2). UV-C induced 2.8- or 1.3 and 2.3- or 0.8 log-reductions of total aerobes or moulds and yeasts in the multi-fruit-ISD and orange-ISD, respectively. Compared to the other models, the Coroller and biphasic models showed a better fit and more accurate parameter estimates. UV-C-induced HMF production was not significant in the ISDs. The current study found that the UV-C treatment was more effective than typical heat pasteurization for inactivating A. acidoterrestris spores in isotonic drinks, following a similar trend for E. coli and native microbiota.