Worldwide, antibiotic usage exceeds 100,000 tons per year and there is increasing concern over the fate of these substances. Antibiotics are ubiquitous in the environment and significant concentrations have been detected in fresh waters. In this review, we highlight important aspects of antibiotic pollution in fresh waters: that concentrations of antibiotics in the environment are substantial, that micro-organisms are susceptible to this, that bacteria can evolve resistance in the environment, and that antibiotic pollution affects natural food webs while interacting with other stressors; which taken together poses a number of challenges for environmental scientists. In the literature, we found examples of considerable antibiotic pollution in fresh waters. In the Americas, antibiotic concentrations of up to 15 μg/L have been measured; with higher concentrations reported from European and African studies (over 10 μg/L and 50 μg/L respectively), and in Asian-pacific countries concentrations over 450 μg/L have been detected. While these concentrations might not be deemed harmful to humans, non-target freshwater organisms could be affected by them. Bioassays show that some of the antibiotics found in surface waters affect microbes at concentrations below 10 μg/L. Among the most potent antibiotics are those that prevail in streams and rivers in these concentrations, such as ciprofloxacin. Sub-lethal concentrations might not kill prokaryotes but contribute to increased bacterial resistance and change the composition of single-celled communities, as demonstrated in laboratory experiments. This has implications for the microbial food web (e.g. interactions among and between bacteria and their protozoan consumers) and by extension, larger organisms and ecosystem health. The fact that the effects of antibiotics are extremely context-dependent represents a challenge, particularly for in vitro research. We suggest future research avenues, taking into account food web experiments, antibiotics interacting with one another (and other stressors) and discuss how these can help to answer multi-layered research questions.