The appropriate pattern of macronutrient distribution for dietary protocols aimed at treating or preventing obesity and its associated cardiovascular diseases is still a controversial topic of discussion. Recommendations considering a specific percentage or range for monounsaturated fatty acids (MUFA) are rare. It was the aim of this study to analyze long-term, randomized, controlled dietary intervention trials and to investigate the effects of MUFA on the biomarkers of obesity and cardiovascular risk factors. Dietary regimens with a high amount of MUFA (>12%) were compared to those with ≤12%. The biomarkers taken into account were weight, waist circumference, fat mass, total cholesterol, LDL cholesterol, HDL cholesterol, triacylglycerols, systolic and diastolic blood pressure, as well as C-reactive protein. A total of 12 studies met the inclusion criteria. Data analysis was performed using the Review Manager 5.0.25 software. Significant differences between high- and low-MUFA protocols could be observed with respect to fat mass [–1.94 kg (confidence interval –3.72, –0.17), p = 0.03], systolic blood pressure [–2.26 mm Hg (confidence interval –4.28, –0.25), p = 0.03] and diastolic blood pressure [–1.15 mm Hg (confidence interval –1.96, –0.34), p = 0.005] favoring the dietary protocols with >12% MUFA. Therefore, MUFA might represent a useful tool in the design of dietary regimens for obesity and cardiovascular disease.