Misuse of pesticides in farming activities leads to contamination of drinking water sources and is responsible for animal and human health problems. The biobeds are practicable option to minimize contamination by pesticides during preparation, use and washing of equipment for pesticide treatments. This research aimed at testing substrate mixtures to optimize biobed efficiency to remove pesticides under the climate of the Yucatan (México). Agricultural soil and 11 mixtures adding vegetable compost, sisal pulp, corn stover and seaweed were tested under controlled conditions. Each biomixture was exposed to a mixture of five pesticides (2,4-diclorophenoxyacetic acid “2,4-D” [1.08 mg cm−3], atrazine [2.50 mg cm−3], carbofuran [0.23 mg cm−3], diazinon [0.34 mg cm−3], and glyphosate [0.36 mg cm−3]) in a period of 41 days. Monitoring of the dissipation of pesticide residues showed that pesticides were quickly dissipated in soil at microcosm level experiment, while at two critical times of 20 and 41 days, all mixtures of substrates (biomixtures) were efficient in dissipation of high concentrations of pesticide in a short time (>99%). Time, biomixture and type of pesticide were shown to be the main parameters influencing pesticide dissipation (P < 0.05). Several other physicochemical parameters of the biomixtures, such as organic matter (OM), lignin, water holding capacity (WHC), and pH, were also significant on pesticide dissipation (P < 0.05), being pH the most significant.