Background: Systematic reviews of the effects of healthcare interventions frequently include non-randomized studies. These are subject to confounding and a range of other biases that are seldom considered in detail when synthesizing and interpreting the results. Our aims were to assess the reliability and usability of a new Cochrane risk of bias (RoB) tool for non-randomized studies of interventions and to determine whether restricting analysis to studies with low or moderate RoB made a material difference to the results of the reviews. Methods and Findings: We selected two systematic reviews of population-based, controlled non-randomized studies of the relationship between the use of thiazolidinediones (TZDs) and cyclooxygenase-2 (COX-2) inhibitors and major cardiovascular events. Two epidemiologists applied the Cochrane RoB tool and made assessments across the seven specified domains of bias for each of 37 component studies. Inter-rater agreement was measured using the weighted Kappa statistic. We grouped studies according to overall RoB and performed statistical pooling for (a) all studies and (b) only studies with low or moderate RoB. Kappa scores across the seven bias domains ranged from 0.50 to 1.0. In the COX-2 inhibitor review, two studies had low overall RoB, 14 had moderate RoB, and five had serious RoB. In the TZD review, six studies had low RoB, four had moderate RoB, four had serious RoB, and two had critical RoB. The pooled odds ratios for myocardial infarction, heart failure, and death for rosiglitazone versus pioglitazone remained significantly elevated when analyses were confined to studies with low or moderate RoB. However, the estimate for myocardial infarction declined from 1.14 (95% CI 1.07–1.24) to 1.06 (95% CI 0.99–1.13) when analysis was confined to studies with low RoB. Estimates of pooled relative risks of cardiovascular events with COX-2 inhibitors compared with no nonsteroidal anti-inflammatory drug changed little when analyses were confined to studies with low or moderate RoB. The exception was a rise in the relative risk associated with ibuprofen from 1.07 (95% CI 0.97–1.18) to 1.14 (95% CI 1.03–1.26). The main limitation of our study was testing the instrument on a narrow range of pharmacoepidemiological studies; we cannot assume our findings extend to a broader range of interventions and settings. Conclusions: The Cochrane RoB tool highlighted a wide range of risks of bias in studies included in two widely cited reviews and had the potential to change the conclusions of the reviews. Systematic reviews that incorporate non-randomized studies of medical interventions should include a detailed assessment of RoB for each included study.