Background: Ca²⁺-signaling controls sperm cell functions necessary for successful fertilization. Multiple endocrine disrupting chemicals have been found to interfere with normal Ca²⁺-signaling in human sperm cells through an activation of the sperm-specific CatSper Ca²⁺-channel, which is vital for normal male fertility. Objectives: We investigated 53 pesticides for their ability to interfere with CatSper mediated Ca²⁺-signaling and function in human sperm cells. Methods: Effects of the pesticides on Ca²⁺-signaling in human sperm cells were evaluated using a Ca²⁺-fluorometric assay. Effects via CatSper were assessed using the specific CatSper inhibitor RU1968. Effects on human sperm function and viability were assessed using an image cytometry-based acrosome reaction assay and the modified Kremer's sperm–mucus penetration assay. Results: 28 of 53 pesticides were found to induce Ca²⁺-signals in human sperm cells at 10 µM. The majority of these 28 active pesticides induced Ca²⁺-signals through CatSper and interfered with subsequent Ca²⁺-signals induced by the two endogenous CatSper ligands progesterone and prostaglandin E₁. Multiple active pesticides were found to affect Ca²⁺-mediated sperm functions and viability at 10 µM. Low nM dose mixtures of the active pesticides alone or in combination with other environmental chemicals were found to significantly induce Ca²⁺-signals and inhibit Ca²⁺-signals induced subsequently by progesterone and prostaglandin E₁. Conclusions: Our results show that pesticides, both alone and in low nM dose mixtures, interfere with normal Ca²⁺-signaling in human sperm cells in vitro in low nM concentrations. Biomonitoring of the active pesticides in relevant matrices such as blood and reproductive fluids is very limited and the effects of real time human pesticide exposure on human sperm cells and fertility thus remains largely unknown. To which extent human pesticide exposure affects the chances of a successful fertilization in humans in vivo needs further research.