The aim of this study was to assess the effect of different concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on selected parameters of equine gametes under controlled in vitro conditions. Assessments covered maturation of oocytes as well as sperm-cell function and structure. In the first part of the experiment, the influence of different concentrations of TCDD (0.32 ng, 3.2 ng, and 32 ng/mL) on in vitro maturation (IVM) of equine oocytes was evaluated. During IVM, 28.38% of the oocytes reached the metaphase II (matured) stage at a TCDD concentration of 0.32 ng/mL and only 5.14% at a concentration of 3.2 ng/mL. In group, with the highest concentration of TCDD in the medium (32 ng), matured oocytes were not observed. In the second part of experiment, the influence of TCDD (0.32 ng, 3.2 ng, and 32 ng/mL) on sperm parameters, such as motility, plasma membrane integrity, acrosome integrity, mitochondrial activity, lipid peroxidation (LPO), apoptosis, and chromatin status, was evaluated. The present study demonstrates that moderate concentrations of TCDD may activate population of motile sperm cells under in vitro conditions with no influence on sperm-cell structure or LPO. Moreover, the modulation of motility function of sperm was confirmed by the detection of the evident increase of cell number with high mitochondrial potential. Dioxins exert negative impact on both female and male reproductive systems. Thus, an overall analysis of both systems will advance scientific knowledge on the impact of toxic agents on the reproductive system.