The use of pesticides in agricultural systems may have deleterious effects on surrounding environments. Aquatic systems are no exception and are increasingly polluted through the leaching of pesticides from agricultural activities. However, the pesticide pollution effects on key aquatic species have not been studied in many regions. In southern Africa, increasing pesticide use associated with macadamia tree Macadamia integrifolia farming presents a growing risk to surrounding aquatic ecosystems. This study assessed behavioural responses of an important and widely-distributed freshwater fish, Mozambique tilapia Oreochromis mossambicus, following exposure to three commonly used macadamia pesticides (i.e., Karate Zeon 10 CS, Mulan 20 SP, Pyrinex 250 CS) at different concentrations (0.7–200 µL, 0.3–1000 mg, and 0.7–8750 µL, respectively) over 24 h. Behavioural responses, i.e., swimming erratically, surfacing, vertical positioning, loss of equilibrium, being motionless and mortality were observed after pesticides exposure. Lethal dose 50 (LD50) values of Karate Zeon 10 CS, Mulan 20 SP and Pyrinex 250 CS were 2.1 µL (per water litre dilution—WLD), 5.2 mg (WLD) and 21.5 µL (WLD), respectively. These concentrations are therefore expressed as a maximal threshold usage in the environment around macadamia farms and a minimum distance of the plantations to water systems should be considered. Further studies should examine effects on other fish species and aquatic invertebrates to inform on pesticide pollution threats and mitigation plans for the region.