The aim of this work was to investigate the changes in aqueous humor cytokine levels after intravitreal injection of aflibercept in diabetic macular edema (DME) and to evaluate the relationship between cytokines modifications and central macular thickness (CMT) and retinal/choroidal vascular changes using structural and functional optical coherence tomography (OCT). Aqueous concentrations of 38 cytokines were measured via multiplex bead assay. In addition, spectral domain OCT and OCT angiography with SSADA software (XR Avanti® AngioVue) were performed at baseline and after intravitreal injections. VEGF, IL-6, IL-5, IL-1β, Eotaxin, GRO, IL-12p40, IL-12p70, IL-1RA, Flt-3L and IP-10 showed a statistically significant decrease through the follow-up (p < 0.05; p < 0.001), while Fraktalkine and GM-CSF significantly increased (p < 0.05). Best corrected visual acuity significantly increased and CMT significantly decreased during follow-up (p < 0.001 and p = 0.013). Superficial capillary plexus and deep capillary plexus density significantly increased (p < 0.001 and p = 0.014). A positive relation was found between GRO, VEGF, Fraktalkine, IP-10, IL-12p70 aqueous humor levels and CMT (p < 0.05; p < 0.001). Aflibercept is a primary anti-VEGF treatment producing a decrease of DME due to the reduction of vascular permeability, nevertheless other inflammatory cytokines showed modification after aflibercept intravitreal injections probably related to edema modification or to an interaction of aflibercept with other inflammatory cytokines.