Microbiologically induced calcite precipitation (MICP) can be used to fill cracks arising in concrete. Alkaliphilic spore-forming bacteria, which can survive extreme pH values and harsh conditions in concrete matrix, are needed for this purpose. In this study, the viability of Bacillus pseudofirmus in sub-zero temperatures and in a concrete matrix containing several types of cement with different pH and metal ion concentrations was investigated. Expanded clay (EC) was used as a carrier for two-component self-healing agent consisting of Bacillus pseudofirmus spores and calcium lactate. The concrete consists of EC, cement, sand and water. Viability of endospores was measured using a standard microbiological dilution-to-extinction method by CFU counting after plating on alkaline nutrient agar. The results showed that after 72 days of incubation at temperatures below 0 °C (-20 °C), the number of viable spores remained almost constant (~10⁷ CFU/g of EC) compared to control specimens stored at the room temperature. That indicates that B. pseudofirmus has a potential to be used in biological self-healing concrete for the Northern Europe region with high number of freeze-thaw cycles. Out of four cement types commonly used in Lithuania, the best survival rate was obtained in a concrete mix using the low alkali white CEM I cement. The number of viable spores after three days of concrete curing varied from 2.34 × 10³ to 6.59 × 10⁴ CFU/g of EC. Thus, an additional coating of EC aggregates is needed to improve the viability of bacteria in the concrete.