Active sea floor hydrothermal systems are modern analogues of ancient volcanogenic massive sulfide deposits. Hydrothermal sulfide chimneys are one of the important components in these systems and are formed by rapid mixing between seawater and metal-rich hydrothermal fluids venting onto the sea floor. Previous models of chimney growth have been built up mainly based on studies including optical petrographic observations and scanning electron microscopy (SEM). The present study, conducted on a sample from the PACMANUS hydrothermal field (Manus basin, Papua New Guinea), reports for the first time the electron backscattered diffraction (EBSD) observations on a modern sea floor chimney, coupled with synchrotron X-ray fluorescence (SXRF) and scanning electron microscopy-backscattered electron (SEM-BSE) imagery to reveal fine-scale and primary microstructures of sphalerite, allowing the reconstruction of crystal growth history. The results show that sphalerite clusters are formed via the coalescence of multiple smaller sphalerite globules. Electron backscattered diffraction images also highlight that each globule includes an inner zone with fine-grained particles (<1 μm) and an outer zone with elongate blade-shaped crystals (length up to 40 μm), in some cases showing branching dendritic habit. Both zones are dominated by sphalerite with minor other sulfides, such as chalcopyrite, pyrite, and wurtzite. The individual globules are interpreted as forming under conditions of supersaturation within high-temperature gradients, for example, during the mixing between high-temperature (e.g., 300°C) hydrothermal fluids and ambient cold seawater. The occurrence of these contrasting inner and outer zones reflects fluctuation between two regimes: bursts of rapid nucleation from supersaturated fluids that occurred almost instantaneously during the initial mixing and the skeletal crystal growth of particular crystal faces from limited nucleation sites where diffusion-limited boundary layers developed around the growing crystals. Those growing globules then coalesced into columnar aggregates. These observations could potentially have important implications for identifying fossil chimneys in ancient ore deposits. Moreover, this study emphasizes the benefits of advanced techniques, such as EBSD and SXRF, in order to characterize sulfides in various hydrothermal chimneys to reveal crystal growth and fluid mixing history and gain further insight into chimney growth processes.