Background Multiple instances of flight-associated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission during long-haul flights have been reported during the COVID-19 pandemic. However, comprehensive investigations of passenger risk behaviours, before, during and after the flight, are scarce. Methods To investigate suspected SARS-CoV-2 transmission during a flight from United Arab Emirates to Australia in July 2020, systematic, repeated polymerase chain reaction (PCR) testing of passengers in hotel quarantine was linked to whole genome sequencing. Epidemiological analyses of in-depth interviews covering behaviours during the flight and activities pre- and post-boarding were used to identify risk factors for infection. Results Seventeen of the 95 passengers from four different travel origins had PCR-confirmed infection yielding indistinguishable genomic sequences. Two of the 17 passengers were symptomatic within 2 days of the flight, and classified as co-primary cases. Seven secondary cases were seated within two rows of the co-primary cases, but five economy passengers seated further away and three business class passengers were also infected (attack rate = 16% [15/93]). In multivariable analysis, being seated within two rows of a primary case [odds ratio (OR) 7.16; 95% confidence interval (CI) 1.66-30.85] and spending more than an hour in the arrival airport (OR 4.96; 95% CI 1.04-23.60) were independent predictors of secondary infection, suggesting travel-associated SARS-CoV-2 transmission likely occurred both during and after the flight. Self-reported increased hand hygiene, frequent aisle walking and using the bathroom on the plane did not independently affect the risk of SARS-CoV-2 acquisition. Conclusions This investigation identified substantial in-flight transmission among passengers seated both within and beyond two rows of the primary cases. Infection of passengers in separate cabin classes also suggests transmission occurred outside the cabin environment, likely at the arrival airport. Recognizing that transmission may occur pre- and post-boarding may inform contact tracing advice and improve efforts to prevent future travel-associated outbreaks.