Propagule pressure is a leading determinant of population establishment. Yet, an experimental understanding of how propagule size and number (two principle parts of propagule pressure) determine establishment success remains incomplete. Theoretical studies suggest that the timing between introduction events, a component of propagule number, can influence establishment success. However, this dynamic has rarely been explored experimentally. Using Escherichia coli engineered with an Allee effect, we investigated how the timing of two introduction events influences establishment. For populations introduced below the Allee threshold, establishment occurred if the time between two introduction events was sufficiently short, with the length of time between events further reduced by reducing growth rate. Interestingly, we observed that as the density of bacteria introduced in one introduction event increased, the time between introduction events that allowed for establishment increased. Using a mathematical model, we provide support that the mechanism behind these trends is the ability of the first population to modify the environment, which can pave the way for establishment of the second population. Our results provide experimental evidence that the temporal distribution of introduction events regulates establishment, furthering our understanding of propagule pressure and may have implications in invasion biology and infectious disease.