Giardia duodenalis is an intestinal parasite which causes diarrhoeal illness in people. Zoonotic subtypes found in livestock may contribute to human disease occurrence through runoff of manure into multi-use surface water. This study investigated temporal associations among selected environmental variables and G. duodenalis occurrence in livestock reservoirs on human giardiasis incidence using data collected in the Waterloo Health Region, Ontario, Canada. The study objectives were to: (1) evaluate associations between human cases and environmental variables between 1 June 2006 and 31 December 2013, and (2) evaluate associations between human cases, environmental variables and livestock reservoirs using a subset of this time series, with both analyses controlling for seasonal and long-term trends. Human disease incidence exhibited a seasonal trend but no annual trend. A Poisson multivariable regression model identified an inverse association with water level lagged by 1 month (IRR = 0.10, 95% CI 0.01, 0.85, P < 0.05). Case crossover analysis found varying associations between lagged variables including livestock reservoirs (1 week), mean air temperature (3 weeks), river water level (1 week) and flow rate (1 week), and precipitation (4 weeks). This study contributes to our understanding of epidemiologic relationships influencing human giardiasis cases in Ontario, Canada.