This paper investigates the effects on the material microstructure of varying the Inter-Layer Cooling Time (ILCT) during the printing process in laser powder bed fusion (L-PBF) multi-laser machines. Despite these machines allowing higher productivity rates compared to single laser machines, they are affected by lower ILCT values, which could be critical for material printability and microstructure. The ILCT values depend both on the process parameter sets and design choices for the parts and play an important role in the Design for Additive Manufacturing approach in L-PBF process. In order to identify the critical range of ILCT for this working condition, an experimental campaign is presented on the nickel-based superalloy Inconel 718, which is widely used for the printing of turbomachinery components. The effect of ILCT on the microstructure of the material is evaluated in terms of porosity and melt pool analysis on printed cylinder specimens, considering ILCT decreasing and increasing in the range of 22 to 2 s. The experimental campaign shows that an ILCT of less than 6 s introduces criticality in the material microstructure. In particular, at an ILCT value of 2 s, widespread keyhole porosity (close to 1‰) and critical and deeper melt pool (about 200 microns depth) are measured. This variation in melt pool shape indicates a change in the powder melting regime and, consequently, modifications of the printability window promoting the expansion of the keyhole region. In addition, specimens with geometry obstructing the heat flow have been studied using the critical ILCT value (2 s) to evaluate the effect of the surface-to-volume ratio. The results show an enhancement of the porosity value (about 3‰), while this effect is limited for the depth of the melt pool.