Digester acclimatization can improve the efficiency of anaerobic digestion (AD), a sustainable process of generating renewable natural gas from biological decomposition of the organic byproducts from biodiesel production. This study examined the impact of acclimatization rate on biogas production and conversion efficiency in laboratory digesters using inocula from a wastewater treatment plant digester and from an agro-industrial waste digester. The results showed that co-digestion of crude glycerol and biodiesel wastewater at high organic loading rates, up to 6.8 g chemical oxygen demand (COD) L-1 day-1, is possible without addition of other substrates or pretreatment. The cumulative biogas production of the digesters using inoculum from the agro-industrial waste digester was statistically greater than that from the digesters using the wastewater treatment plant digester, indicating that an inoculum with similar physical characteristics may be most appropriate for AD of biodiesel waste. Of the two acclimatization rates studied, the slower rate improved the cumulative biogas production for both inocula. In addition, both inocula exhibited higher maximum daily efficiencies with a slower rate of acclimatization, up to a maximum average daily biogas yield of 621 mL biogas g-1 COD added. This implied that the rate of acclimatization can impact the ability of the digester to efficiently adapt to a new substrate.