Background: Our objective was to determine the effect of fatty acids (FAs) in serum and follicular fluid (FF) on fertilization and intracytoplasmic sperm injection (ICSI) outcomes. Methods: One hundred five women aged 18–38 years undergoing ICSI were recruited in this prospective cohort study. oocyte and emberyo quality was morphologically assessed. FAs in serum and FF were analyzed, using gas chromatography–mass spectrometry (GC-MS). Results: The mean number of mature oocytes was associated with serum levels of oleic acid (r = 0.58; P = 0.002). There were negative correlations between metaphase II oocytes and FF levels of stearic acid (r = −0.19; P = 0.04) and linolenic acid (r = −0.37; P = 0.004). According to the obtained Spearman’s correlation coefficients, serum levels of stearic, palmitoleic and tricosanoic acids were positively correlated with the percent of germinal vesicle (GV) stage oocyte. The mean serum level of eicosapentaenoic acid was significantly higher in pregnant women than in non-pregnant patients (P = 0.006). Good quality embryos’ percentages were negatively correlated with the concentrations of palmitic acid (r = −0.22; P = 0.02). After adjusting the effects of body mass index and age, total FAs were found to have a significant effect on the odds of having high-quality oocytes (percentage of oocytes > 80%; odds ratio =2.55; P = 0.054). Conclusion: Particular FAs affect oocyte maturation and implantation. Apparently, while higher FF levels of saturated FAs, especially palmitic and stearic acids, observed in some metabolic contexts have harmful effects on oocyte maturation and implantation, such effects can be counteracted and developmental competence can be enhanced (at least in vitro) by the presence of unsaturated FAs, e.g. oleic and eicosapentaenoic acids.