Background: We estimated the effectiveness of seasonal inactivated influenza vaccine and the potential influence of timing of immunization on vaccine effectiveness (VE) using data from the 2016 southern hemisphere influenza season. Methods: Data were pooled from three routine syndromic sentinel surveillance systems in general practices in Australia. Each system routinely collected specimens for influenza testing from patients presenting with influenza-like illness. Next generation sequencing was used to characterize viruses. Using a test-negative design, VE was estimated based on the odds of vaccination among influenza-positive cases as compared to influenza-negative controls. Subgroup analyses were used to estimate VE by type, subtype and lineage, as well as age group and time between vaccination and symptom onset. Results: A total of 1085 patients tested for influenza in 2016 were included in the analysis, of whom 447 (41%) tested positive for influenza. The majority of detections were influenza A/H3N2 (74%). One-third (31%) of patients received the 2016 southern hemisphere formulation influenza vaccine. Overall, VE was estimated at 40% (95% CI: 18–56%). VE estimates were highest for patients immunized within two months prior to symptom onset (VE: 60%; 95% CI: 26–78%) and lowest for patients immunized >4 months prior to symptom onset (VE: 19%; 95% CI: −73–62%). Discussion: Overall, the 2016 influenza vaccine showed good protection against laboratory-confirmed infection among general practice patients. Results by duration of vaccination suggest a significant decline in effectiveness during the 2016 influenza season, indicating immunization close to influenza season offered optimal protection.